Carotenoids (e.g., lycopene, β-carotene, zeaxanthin, canthaxanthin and astaxanthin) represent one of the most widely distributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep red color. Eye-catching examples of carotenogenic tissues include carrots, tomatoes, red peppers, and the petals of daffodils and marigolds. Carotenoids are synthesized by all photosynthetic organisms, as well as some bacteria and fungi. These pigments have important functions in photosynthesis, nutrition, and protection against photooxidative damage; as such, they are used today in e.g., food ingredients/colors, animal feed ingredients, pharmaceuticals, cosmetics and as nutritional supplements.
Animals do not have the ability to synthesize carotenoids but must obtain these nutritionally important compounds through their dietary sources. Many animals exhibit an increase in tissue pigmentation when carotenoids are included in their diets, a characteristic often valued by consumers. For example, canthaxanthin and astaxanthin are commonly used in commercial aquaculture industries to pigment shrimp and salmonid fish. It has also been reported that astaxanthin may be a dietary requirement for the growth and survival of some salmonid species (Christiansen et al., Aquaculture Nutrition, 1:189-198 (1995)). Similarly, lutein, canthaxanthin and astaxanthin are commonly used as pigments in poultry feeds to increase the pigmentation of chicken skin and egg yolks.
Industrially, only a few carotenoids are used, despite the existence of more than 600 different carotenoids identified in nature. This is largely due to difficulties in production and high associated costs. For example, the predominant source of aquaculture pigments used in the market today are produced synthetically and are sold under such trade names as Carophyll® Red (canthaxanthin; DSM Nutritional Products, Heerlen, NL) and Carophyll® Pink (astaxanthin; DSM Nutritional Products); however, the cost of utilizing the synthetically produced pigments is quite high even though the amount of pigment incorporated into the fishmeal is typically less than 100 ppm.
A further concern associated with the use of synthetically produced carotenoids for feed formulations is the common addition of synthetic antioxidants to act as a preservative and to help protect the pigments from oxidation. Many of these synthetic antioxidants are facing significant questions concerning their safety; for example, many adverse health effects (e.g., carcinogenesis, cytotoxicity, etc.) have been reported with the use of ethoxyquin [6-ethoxy-2,2,4-trimethyl-1H-quinoline] (Blaszczyk et. al., Cell Mol. Biol. Lett., 10 (1):15-21 (2005); Blaszczyk et al., Mutat. Res., 542:117-128 (2003); Little, A. D., Chemical Evaluation Committee Draft Report, Ethoxyquin, CAS Number 91-53-2, submitted to National Toxicology Program, Executive Summary of Safety and Toxicity Information, U.S. Department of Health and Human Services, (1990)). As such, there is a need for a source of natural carotenoids that optionally comprise natural antioxidants suitable for their stabilization.
Natural carotenoids can either be obtained by extraction of plant material or by microbial synthesis; but, only a few plants are widely used for commercial carotenoid production and the productivity of carotenoid synthesis in these plants is relatively low. Microbial production of carotenoids is a more attractive production route. Examples of carotenoid-producing microorganisms include: algae (Haematococcus pluvialis, sold under the tradename NatuRose™ (Cyanotech Corp., Kailua-Kona, Hi.; Dunaliella sp.), yeast (Phaffia rhodozyma, recently renamed as Xanthophyllomyces dendrorhous; Thraustochytrium sp.; Labyrinthula sp.; and Saccharomyces cerevisiae), and bacteria (Paracoccus marcusii, Bradyrhizobium, Rhodobacter sp., Brevibacterium, Escherichia coli and Methylomonas sp.). Additionally, recombinant production of carotenoids is also possible, since the genes involved in carotenoid biosynthesis are well-known and have been heterologously expressed in a variety of host cells (e.g., E. coli, Candida utilis, Saccharomyces cerevisiae, Methylomonas sp.). Thus far, few of these demonstrations are suitable to produce a carotenoid product in significant quantities in a cost-effective manner for industrial use.
Many commercial products are formulated to contain a mixture of pigments and fats/lipids and/or natural antioxidants. For example, animal feeds, dietary supplements, and personal care products are often formulated to contain carotenoids, polyunsaturated fatty acids (PUFAs) and antioxidants (e.g., CoQ10). For fish feed formulations, fish oil is often supplemented to the feed to provide the necessary caloric intake and to provide essential fatty acids such as the ω-3/ω-6 PUFAs. Typically, for example, a commercial product formulator will obtain these compounds from a variety of sources and formulate them into a final product that contains an effective amount of each ingredient. The composition, purity and source of each ingredient may vary, resulting in a final product formulation that may require significant monitoring and/or processing to obtain the desired product specifications.
Engineering a microorganism to simultaneously produce carotenoids and fats/lipids and/or an additional antioxidant could create a higher value product and prove advantageous for commercial production economics (and therefore availability) to consumers. One class of organisms that are especially suitable as a production platform for synthesis of pigmented microbial oils (optionally comprising PUFAs and/or additional antioxidants) are the oleaginous yeast. Oleaginous yeast are defined as those yeast that are naturally capable of oil synthesis and accumulation, wherein oil accumulation is at least 25% of the cellular dry weight. In particular, Yarrowia lipolytica is an oleaginous yeast that has a number of characteristics that make it particularly useful for the production of ω-3/ω-6 PUFAs (see for example commonly owned U.S. Pat. No. 7,238,482 and U.S. patent application Ser. No. 11/198,975, Ser. No. 11/265,761, Ser. No. 11/264,784 and Ser. No. 11/264,737, corresponding to PCT Publication Nos. WO 2006/033723, WO 2006/052870, WO 2006/055322 and WO 2006/052871, respectively; see also U.S. Patent Application No. 60/977,177).
PCT Publication No. WO 2006/102342 (Microbia, Inc.) has engineered an oleaginous yeast to produce carotenoids, thereby resulting in a pigmented microbial product. In particular, they demonstrated greater than 4 mg carotene per gram dry cell weight in Yarrowia lipolytica (see Example 2 therein). However, there have been no previous reports of an oleaginous microbial host cell that can co-produce carotenoids and ω-3/ω-6 PUFAs, to thereby result in a single product comprising both ingredients. This is particularly attractive when the recombinant cell biomass is used directly in the formulation (e.g., as an animal feed).
The problem to be solved therefore, is to provide a recombinant oleaginous yeast capable of producing at least one carotenoid. A further problem to be solved is to provide an oleaginous yeast capable of producing at least one carotenoid in combination with an ω-3/ω-6 PUFA and/or at least one additional antioxidant.